There has been increasing evidence supporting a role of inflammation in impaired insulin action in peripheral tissues in both humans and animal models of insulin resistance and type 2 diabetes. CAP (Cbl associated protein), a ubiquitously expressed adapter protein whose expression has been described in both adipose and skeletal muscle tissues, is believed to act in concert with Cbl (the Cbl/CAP pathway) to stimulate glucose uptake in peripheral insulin sensitive tissues such as skeletal muscles and adipose tissue. The Cbl/CAP pathway has also been shown to be involved in the proliferation and motility of macrophages, which are recruited to these tissues in response to inflammatory stimuli.
Data from cultured adipocytes support a role of a Cbl/CAP complex in insulin stimulated glucose uptake. Transfection of 3T3-L1 adipocytes with a non-functional CAP results in a near complete inhibition of GLUT4 translocation and glucose uptake (Chiang, S. H., et al., Nature 410: 944-948, 2001; Liu, J., et al., J. Biol. Chem. 278: 36754-36762, 2003). However, in 3T3-L1 adipocytes siRNA silencing of Cbl, CAP or Crkll was ineffective at diminishing glucose uptake in response to insulin stimulation (Mitra, P. et al., J. Biol. Chem. 279: 37431-37435, 2004). Thirone, A. C., et al., (Endocrinology 145: 281-93, 2004) described higher expression of Cbl in adipose tissue compared to muscle in rats with no detectable CAP in muscle homogenates. Wadley, G. D., et al. (Diabetologia 47: 412-419, 2004) demonstrated CAP expression in skeletal muscle, but described higher levels of CAP expression in 3T3-L1 adipocytes than in soleus muscle from Zucker rats. Despite these decrepancies in tissue specific CAP expression, the protein content and phosphorylation state appears to be sensitive to alterations in whole body insulin sensitivity. For example, basal skeletal muscle Cbl phosphorylation can be increased in insulin-resistant rats relative to lean controls (Wadley, G. D., et al. Diabetologia 47: 412-419, 2004). Likewise, in adipose tissue, induction of insulin-resistance in rats can result in increased expression of both Cbl and CAP, as well as an increase in both their association and phosphorylation in response to insulin stimulation.
In addition to activation of pathways downstream of receptor tyrosine kinases (RTKs), Cbl has also been shown to exert a dampening role by stimulating the ubiquitination and subsequent degradation of the ligand-bound RTKs (Duan, L., et al., Immunity 21: 7-17, 2004). This dual and opposing role of Cbl can be illustrated by the actions of Cbl downstream of the activated RTK, colony stimulating factor-1 (CSF-1). Lee, P. S., et al. (EMBO J. 18: 3616-3628 1999) demonstrated that Cbl activity negatively regulated macrophage proliferation via receptor ubitiquination. Activation of Cbl in macrophages can also control proliferation, survival, differentiation, adhesion and cell motility via downstream Crkll activation (Erdreich-Epstein, A., et al., J. Leukoc. Biol. 65:523-534, 1999; Caveggion, E., et al., J. Cell Physiol. 195: 276-89, 2003; Husson, H. et al., Oncogene 14: 2331-2338, 1997; Wang, Y., et al., J. Cell Biochem. 72: 119-134).
Macrophage infiltration of adipose tissue and intramuscular fat is known to occur in obesity (Weisberg, S. P., et al., J. Clin. Invest. 112: 1796-1808, 2003; Xu, H., et al., J. Clin. Invest. 112: 1821-1830, 2003). In addition, tissue levels of inflammatory markers such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1) and inducible nitric oxide synthase (iNOS) are also elevated in obesity and are believed to be derived from the infiltrating macrophages (Weisberg, S. P., et al., J. Clin. Invest. 112: 1796-1808, 2003). Moreover, increased expression of inflammatory genes can be evident as early as 3 weeks after the introduction of a high fat diet, with worsening severity at the onset of hyperinsulinemia (Xu, H., et al., J. Clin. Invest. 112: 1821-1830, 2003). However, previous studies do not reveal if the adaptor protein, CAP, is present and/or essential to normal inflammatory activity of macrophages, nor do they disclose therapies involving inhibition of CAP expression or activity